1. Field of the Invention
The invention relates to a process for producing a highly purified sweetener from the extract of the Stevia rebaudiana Bertoni plant and its use in various food products and beverages.
2. Description of the Related Art
At present, artificial sweeteners such as dulcin, sodium cyclamate and saccharin have been banned or restricted in view of food sanitation, and harmless natural sweeteners are receiving ever-increasing demand. The sweet herb Stevia rebaudiana Bertoni, produces an alternative sweetener with the added advantage that Stevia sweeteners are natural plant products. In addition, the sweet steviol glycosides have functional and sensory properties superior to those of many high potency sweeteners.
The sweet diterpene glycosides of Stevia have been characterized and eight sweet glycosides of steviol have been identified. These glycosides accumulate in Stevia leaves where they may be present at from 10 to 20% of the leaf dry weight. On a dry weight basis, a typical profile for the four major glycosides found in the leaves of Stevia includes 0.3% dulcoside, 0.6% rebaudioside C, 3.8% rebaudioside A and 9.1% stevioside. Other glycosides identified within Stevia include rebaudiosides B, C, and E, and dulcosides A and B. Rebaudioside B may be an artifact formed from rebaudioside A during extraction since both rebaudioside A and rebaudioside D are found to convert to rebaudioside B by alkaline hydrolysis.
Of the four major diterpene glycoside sweeteners present in Stevia leaves only two, stevioside and rebaudioside A, have had their physical and sensory properties well characterized. Stevioside and rebaudioside A were tested for stability in carbonated beverages and found to be both heat and pH stable (Chang and Cook, 1983). Stevioside is between 110 and 270 times sweeter than sucrose, rebaudioside A between 150 and 320 times sweeter than sucrose, and rebaudioside C between 40 and 60 times sweeter than sucrose. Dulcoside A was 30 times sweeter than sucrose. Rebaudioside A was the least astringent, the least bitter, had the least persistent aftertaste and was judged to have the most favorable sensory attributes of the four major steviol glycosides (Phillips, 1989 and Tanaka, 1997).
Methods for the recovery of diterpene glycosides from the Stevia rebaudiana plant using water or large amounts of organic solvents, such as methanol and ethanol are described in U.S. Pat. No. 4,361,697, U.S. Pat. No. 4,082,858, U.S. Pat. No. 4,892,938, U.S. Pat. No. 5,972,120, and U.S. Pat. No. 5,962,678.
However, stevioside possesses residual bitterness and aftertaste, which affect its qualitative characteristics. They can be eliminated by the reaction of intermolecular transglycosylation of various enzymes, upon which the attachment of new carbohydrates at positions C13 and C19 takes place. It is the number of carbohydrate units in the above-mentioned positions that determines the quality and degree of the component's sweetness.
Pullulanase, isomaltase (Lobov et al., 1991), β-galactosidase (Kitahate et al., 1989), and dextrine saccharase (Yamamoto et al., 1994) are used as transglycosylating enzymes, with pullulan, maltose, lactose, and partially hydrolyzed starch, respectively, being donors.
The treatment with pullulanase results in production of 13-O-[β-maltotriosyl-(1,2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol; 13-O-[β-maltosyl-(1,2)-β-glucosyl]-19-O-β-D-glucosyl-steviol and 13-O-[β-sephorosyl-19-O-β-maltotriosyl-steviol. Although the yields of the transglycosylated products were rather low, the selectivity in terms of the yield of the desirable mono- and di-derivatives was higher than in the case of CGTase (Lobov et al., 1991).
In case of maltase, three transglycosylated products are also produced, namely 13-O-[β-sephorosyl-19-O-β-isomaltosyl-steviol; 13-O-[β-isomaltosyl-(1,2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol and 13-O-[β-nigerosyl-(1,2)-β-D-glucosyl]-19-O-β-D-glucosyl-steviol.
The transglucosylation of stevioside was also done by action of cyclodextrin glucanotransferases (CGTase) produced by Bacillus stearothermophilus FERM-P No 2222 (U.S. Pat. No. 4,219,571).
However, all the abovementioned processes are based on the modification either of stevioside or rebaudioside A, which have to be isolated before such treatment. The sweetness level of the products obtained ranges between 50-120, and the final products contains about 20% of maltooligosacharides that decrease the healthy properties of the product. Besides that, the reactions conditions were not defined clearly (U.S. Pat. No. 4,219,571). For example, the concentration range of stevioside (from 0.1% to 20%) and enzyme (CGTase) (from 1% to 50%), and also the limits of temperature (from 20° C. to 80° C.), pH (from 3 to 10), and transformation duration (from 5 to 80 hours) are too large. Thus, it is impossible to define the preferred embodiment.
On the other hand, defining the optimum reaction condition can considerably decrease the reaction time and consumption of enzyme, and also increase the yield of the final product. It can maximize the economic contribution of the industrial process. Besides, the filtration rate of the reaction mixture, obtained at low temperatures, after treatment by activated carbon is very low, which can be incommensurably improved by increasing the reaction temperature. It also helps to decrease the non-modified amounts of glycosides.
Accordingly, it is desirable to provide a process for the simultaneous enzymatic modification of glycosides, existing in the commercial extract of Stevia rebaudiana Bertoni plant, for manufacturing a purified sweetener with high sweetness, and for use thereof in various beverages and food products.